Prothrombin fractionation

The term "euglobulin" has not always been used in this sense frequently it has been used to denote proteins precipitated by 0.33 saturated amme um sulfate solution. The two definitions are far from identical, as will become apparent in the subsequent discusnon. Moreover, some authors have denoted as euglobulins only those proteins precipitated by removal of salt at some particular pH—commonly at or near pH 7. This definition is much more restricted than that adopted here the prothrombin fraction of plasma, for instance, is very insoluble at low ionic strength at pH 6, but is soluble in the complete absence of salt at pH 7. Wh shall consistently term such proteins "euglobulins in the present discussion. 

Table 9.12 Clotting time of blood from a calf affected by sweet clover disease when treated with the prothrombin fraction from a normal animal or an affected animal...

The clear serum of this example is an amber liquid free from prothrombin, thrombin, fibrinogen and fibrin. It contains profibrinolysin and is excellently suited to further purification by salt precipitation fractionation, as given below. 

Ferric ion was immobilized on a Chelating Sepharose Fast Flow column preparatory to the separation of seven enkephalin-related phosphopep-tides.17 Non-phosphorylated peptides flowed through the column, and the bound fraction contained the product. The capacity of the column was found to be 23 pmol/mL by frontal elution analysis. Cupric ion was immobilized on Chelating Superose for the isolation of bovine serum albumin.18 Cupric ion was immobilized on a Pharmacia HiTrap column for the separation of Protein C from prothrombin, a separation that was used to model the subsequent apparently successful separation of Factor IX from prothrombin Factor IX activity of the eluate was, however, not checked.19 Imidazole was used as the displacement agent to recover p-galactosidase from unclarified homogenates injected onto a nickel-loaded IMAC column.20 Pretreatment with nucleases and cleaning in place between injections were required procedures. A sixfold purification factor was observed. 

CBC, complete blood cell count EEG, electroencephalogram NSAIDs, nonsteroidal antiinflammatory drugs O2, oxygen PPF, plasma protein fraction PT, prothrombin time TIPS, transjugular intrahepatic portosystemic shunt. 

Morrissey 53) used transmission infrared spectroscopy to study protein adsorption onto silica particles in a heavy water (DzO) buffer. By observing the shift in the amide I absorption band, he could deduce the fraction of protein carbonyl groups involved in bonding to the silica surface. He found that bovine IgG had a bound fraction of 0.20 at low bulk solution concentrations, but only about 0.02 at high solution concentrations. However, neither prothrombin nor bovine serum albumin exhibited a change in bound fraction with concentration. Parallel experiments with flat silica plates using ellipsometry showed that the IgG-adsorbed layers had an optical thickness of 140 A and a surface concentration of 1.7 mg/m2 at low bulk solution concentration — in concentrated solutions the surface amount was 3.4 mg/m2 with a thickness of 320 A (Fig. 17). 

Brummelhuis HGJ (1980) Preparation of the Prothrombin complex. In Curling JM (ed) Methods of plasma protein fractionation. Academic Press, New York, p 117... 

Proteins used in this study are albumin (bovine, crystalline, Nutri-tritional Biochemical Co.), y-globulin (bovine. Fraction II, Nutritional Biochemical Co.), prothrombin (bovine. Fraction III-2, Nutritional Biochemical Co.), I-y-globulin and I-fibrinogen (New England Nuclear Co.), and I-albumin (Squibb Co.). 

Vitamin K is critical to (he formation of clotting factors VII. IX. and X. These factors are glycoproteins that h.avc y-vaiboxyglutamic acid residues at the N-terminal end of (he ptptide chain. The enzyme involved in forming an active prothrombin is a vitamin K-dcpendcn( carboxyla.se located in the microsomal fraction of liver cells. It has been sug-... 

Although there is an explosive increase in the reaction rate, only a small fraction (< Cl%) of the total prothrombin available ( 1.5 /zM) is converted to thrombin. The concentration of thrombin required to convert fibrinogen... 

Sertraline has been found to produce small increases in the free fraction of warfarin and a modest (-9%) increase in prothrombin time, which are considered clinically insignificant. Of the SSRIs, sertraline is assumed to exert the... 

Here, a laser beam totally internally reflects at a sohd/hquid interface, creating an evanescent field, which penetrates only a fraction of the wavelength into the liquid domain. When using planar phosphoHpid bilayer and fluorescently labeled proteins, this method allows the determination of adsorption/desorption rate constants and surface diffusion constants [171—173]. Figure 6.29 shows a representative TIRF-FPR curve for fluorescein-labeled prothrombin bound to planar membranes. In this experiment the experimental conditions are chosen such that the recovery curve is characterized by the prothrombin desorption rate. It should be mentioned that, similar to other applications of fluorescence microscopy, two and three photon absorption might be combined with FRAP in the near future. 

The interaction of heparin with human plasma low-density lipoprotein has been studied by gel permeation chromatography A fraction containing degradation products of fibrinogen formed in blood plasma after injection of thrombin also contains a complex of fibrinogen and heparin. The rate of inactivation of thrombin and Factor by anti-thrombin has been shown to be directly proportional to the chain-length of heparin.The effects of heparin on the reactions catalysed by Factor have been examined.Heparin inhibits the ability of phospholipid to accelerate prothrombin activation in a mechanism which appears to involve the displacement of Factor X from the surface of the phospholipid. 

Prothrombin, the precursor of thrombin, is present in plasma only in small amounts (0.2% of the protein content of plasma) and can be purified by acid precipitation by adsorption on magnesium hydroxide and fractionation with ammonium sulfate. The purified protein contains glucose (6.5%), neuraminic acid (5%), and unidentified amino sugars. A study of the amino acid composition reveals that the molecule contains 19 different amino acids. Prothrombin has only one N-terminal amino acid (alanine), and no carboxy C-terminal can be detected by hydrolyzing the molecule with carboxypeptidase A or B. Prothrombin is an ellipsoid molecule (119 A x 34 A) with a molecular weight of 62,700. On electrophoresis of plasma, prothrombin migrates between the a2- and the jS-globulin. 

This fraction contains virtually all the antibodies of plasma, as well as prothrombin, the isoagglutinins, plasminogen, and the component C, of complement. The separation of these various components is, of course, of major importance, and a systematic procedure has been developed by Oncley, Melin, Cameron, Richert and Gross (161). Briefly, it is based on the following observations ... 

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